Corn Event DAS-59122-7 and Methods for Detection Thereof

ABSTRACT

The invention provides DNA compositions that relate to transgenic insect resistant maize plants. Also provided are assays for detecting the presence of the maize DAS- 59122 - 7  event based on the DNA sequence of the recombinant construct inserted into the maize genome and the DNA sequences flanking the insertion site. Kits and conditions useful in conducting the assays are provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. Ser. No. 11/237,222 filed Sep.28, 2005, which claims the benefit of U.S. Provisional Application Ser.No. 60/614,225, filed Sep. 29, 2004, the contents of which are hereinincorporated by reference in their entirety.

FIELD OF INVENTION

Embodiments of the present invention relate to the field of plantmolecular biology, specifically an embodiment of the invention relatesto a DNA construct for conferring insect resistance to a plant.Embodiments of the invention more specifically relate to an insectresistant corn plant DAS-59122-7 and to assays for detecting thepresence of corn plant DAS-59122-7 DNA in a sample and compositionsthereof.

BACKGROUND OF INVENTION

An embodiment of this invention relates to the insect resistant corn(Zea mays) plant DAS-59122-7, also referred to as maize line DAS-59122-7or maize event DAS-59122-7, and to the DNA plant expression construct ofcorn plant DAS-59122-7 and the detection of the transgene/flankinginsertion region in corn plant DAS-59122-7 and progeny thereof.

Corn is an important crop and is a primary food source in many areas ofthe world. Damage caused by insect pests is a major factor in the lossof the world's corn crops, despite the use of protective measures suchas chemical pesticides. In view of this, insect resistance has beengenetically engineered into crops such as corn in order to controlinsect damage and to reduce the need for traditional chemicalpesticides. One group of genes which have been utilized for theproduction of transgenic insect resistant crops are the delta-endotoxinsfrom Bacillus thuringiensis (B.t.). Delta-endotoxins have beensuccessfully expressed in crop plants such as cotton, potatoes, rice,sunflower, as well as corn, and have proven to provide excellent controlover insect pests. (Perlak, F. J et al. (1990) Bio/Technology 8,939-943; Perlak, F. J. et al. (1993) Plant Mol. Biol. 22: 313-321;Fujimoto H. et al. (1993) Bio/Technology 11: 1151-1155; Tu et al. (2000)Nature Biotechnology 18:1101-1104; PCT publication number WO 01/13731;and Bing J W et al. (2000) Efficacy of Cry1F Transgenic Maize, 14^(th)Biennial International Plant Resistance to Insects Workshop, FortCollins, Colo.).

The expression of foreign genes in plants is known to be influenced bytheir location in the plant genome, perhaps due to chromatin structure(e.g., heterochromatin) or the proximity of transcriptional regulatoryelements (e.g., enhancers) close to the integration site (Weising etal., Ann. Rev. Genet 22:421-477, 1988). At the same time the presence ofthe transgene at different locations in the genome will influence theoverall phenotype of the plant in different ways. For this reason, it isoften necessary to screen a large number of events in order to identifyan event characterized by optimal expression of an introduced gene ofinterest. For example, it has been observed in plants and in otherorganisms that there may be a wide variation in levels of expression ofan introduced gene among events. There may also be differences inspatial or temporal patterns of expression, for example, differences inthe relative expression of a transgene in various plant tissues, thatmay not correspond to the patterns expected from transcriptionalregulatory elements present in the introduced gene construct. For thisreason, it is common to produce hundreds to thousands of differentevents and screen those events for a single event that has desiredtransgene expression levels and patterns for commercial purposes. Anevent that has desired levels or patterns of transgene expression isuseful for introgressing the transgene into other genetic backgrounds bysexual outcrossing using conventional breeding methods. Progeny of suchcrosses maintain the transgene expression characteristics of theoriginal transformant. This strategy is used to ensure reliable geneexpression in a number of varieties that are well adapted to localgrowing conditions.

It would be advantageous to be able to detect the presence of aparticular event in order to determine whether progeny of a sexual crosscontain a transgene of interest. In addition, a method for detecting aparticular event would be helpful for complying with regulationsrequiring the pre-market approval and labeling of foods derived fromrecombinant crop plants, for example, or for use in environmentalmonitoring, monitoring traits in crops in the field, or monitoringproducts derived from a crop harvest, as well as for use in ensuringcompliance of parties subject to regulatory or contractual terms.

It is possible to detect the presence of a transgene by any nucleic aciddetection method known in the art including, but not limited to, thepolymerase chain reaction (PCR) or DNA hybridization using nucleic acidprobes. These detection methods generally focus on frequently usedgenetic elements, such as promoters, terminators, marker genes, etc.,because for many DNA constructs, the coding region is interchangeable.As a result, such methods may not be useful for discriminating betweendifferent events, particularly those produced using the same DNAconstruct or very similar constructs unless the DNA sequence of theflanking DNA adjacent to the inserted heterologous DNA is known. Forexample, an event-specific PCR assay is described in U.S. Pat. No.6,395,485 for the detection of elite event GAT-ZM1. Accordingly, itwould be desirable to have a simple and discriminative method for theidentification of event DAS-59122-7.

SUMMARY OF INVENTION

Embodiments of this invention relate to methods for producing andselecting an insect resistant monocot crop plant. More specifically, aDNA construct is provided that when expressed in plant cells and plantsconfers resistance to insects. According to one aspect of the invention,a DNA construct, capable of introduction into and replication in a hostcell, is provided that when expressed in plant cells and plants confersinsect resistance to the plant cells and plants. The DNA construct iscomprised of a DNA molecule named PHI17662A and it includes three (3)transgene expression cassettes. The first expression cassette comprisesa DNA molecule which includes the promoter, 5′ untranslated exon, andfirst intron of the maize ubiquitin (Ubi-1) gene (Christensen et al.(1992) Plant Mol. Biol. 18:675-689 and Christensen and Quail (1996)Transgenic Res. 5:213-218) operably connected to a DNA molecule encodinga B.t. δ-endotoxin identified as Cry34Ab1 (U.S. Pat. Nos. 6,127,180,6,624,145 and 6,340,593) operably connected to a DNA molecule comprisinga Pin II transcriptional terminator isolated from potato (Gyheung An etal. (1989) Plant Cell. 1:115-122). The second transgene expressioncassette of the DNA construct comprises a DNA molecule encoding thewheat peroxidase promoter (Hertig et al (1991) Plant Mol. Biol.16:171-174) operably connected to a DNA molecule encoding a B.t.δ-endotoxin identified as Cry35Ab1 (U.S. Pat. Nos. 6,083,499, 6,548,291and 6,340,593) operably connected to a DNA molecule comprising a Pin IItranscriptional terminator isolated from potato (Gyheung An et al.(1989) Plant Cell. 1:115-122). The third transgene expression cassetteof the DNA construct comprises a DNA molecule of the cauliflower mosaicvirus (CaMV) 35S promoter (Odell J. T. et al. (1985) Nature 313:810-812; Mitsuhara et al. (1996) Plant Cell Physiol. 37: 49-59) operablyconnected to a DNA molecule encoding a phosphinothricinacetyltransferase (PAT) gene (Wohlleben W. et al. (1988) Gene 70: 25-37)operably connected to a DNA molecule comprising a 3′ transcriptionalterminator from (CaMV) 35S (see Mitsuhara et al. (1996) Plant CellPhysiol. 37: 49-59). Plants containing the DNA construct are alsoprovided.

According to another embodiment of the invention, compositions andmethods are provided for identifying a novel corn plant designatedDAS-59122-7, which methods are based on primers or probes whichspecifically recognize the 5′ and/or 3′ flanking sequence ofDAS-59122-7. DNA molecules are provided that comprise primer sequencesthat when utilized in a PCR reaction will produce amplicons unique tothe transgenic event DAS-59122-7. These molecules may be selected fromthe group consisting of:

-   -   5′-GTGGCTCCTTCAACGTTGCGGTTCTGTC-3′ (SEQ ID NO: 1);    -   5′-CGTGCAAGCGCTCAATTCGCCCTATAGTG-3′ (SEQ ID NO: 2);    -   5′-AATTGAGCGCTTGCACGTTT-3′ (SEQ ID NO: 3);    -   5′-AACAACAAGACCGGCCACACCCTC-3′ (SEQ ID NO: 4);    -   5′-GAGGTGGTCTGGATGGTGTAGGTCA-3′ (SEQ ID NO: 5);    -   5′-TACAACCTCAAGTGGTTCCTCTTCCCGA-3′ (SEQ ID NO: 6);    -   5′-GAGGTCTGGATCTGCATGATGCGGA-3′ (SEQ ID NO: 7);    -   5′-AACCCTTAGTATGTATTTGTATT-3′ (SEQ ID NO: 8);    -   5′-CTCCTTCAACGTTGCGGTTCTGTCAG-3′ (SEQ ID NO: 9);    -   5′-TTTTGCAAAGCGAACGATTCAGATG-3′ (SEQ ID NO: 10);    -   5′-GCGGGACAAGCCGTTTTACGTTT-3′ (SEQ ID NO: 11);    -   5′-GACGGGTGATTTATTTGATCTGCAC-3′ (SEQ ID NO: 12);    -   5′-CATCTGAATCGTTCGCTTTGCAAAA-3′ (SEQ ID NO: 13);    -   5′-CTACGTTCCAATGGAGCTCGACTGTC-3′ (SEQ ID NO: 14);    -   5′-GGTCAAGTGGACACTTGGTCACTCA-3′ (SEQ ID NO: 15);    -   5′-GAGTGAAGAGATAAGCAAGTCAAAG-3′ (SEQ ID NO: 16);    -   5′ -CATGTATACGTAAGTTTGGTGCTGG-3′ (SEQ ID NO: 17);    -   5′-AATCCACAAGATTGGAGCAAACGAC-3′ (SEQ ID NO: 18)    -   5′-CGTATTACAATCGTACGCAATTCAG-3′ (SEQ ID NO: 36);    -   5′-GGATAAACAAACGGGACCATAGAAG-3′ (SEQ ID NO: 37) and complements        thereof.        The corn plant and seed comprising these molecules is an        embodiment of this invention. Further, kits utilizing these        primer sequences for the identification of the DAS-59122-7 event        are provided.

An additional embodiment of the invention relates to the specificflanking sequences of DAS-59122-7 described herein, which can be used todevelop specific identification methods for DAS-59122-7 in biologicalsamples. More particularly, the invention relates to the 5′ and/or 3′flanking regions of DAS-59122-7, SEQ ID NO: 19, 5′ flanking and SEQ IDNO: 20, 3′ flanking, respectively, which can be used for the developmentof specific primers and probes. A further embodiment of the inventionrelates to identification methods for the presence of DAS-59122-7 inbiological samples based on the use of such specific primers or probes.

According to another embodiment of the invention, methods of detectingthe presence of DNA corresponding to the corn event DAS-59122-7 in asample are provided. Such methods comprise: (a) contacting the samplecomprising DNA with a DNA primer set, that when used in a nucleic acidamplification reaction with genomic DNA extracted from corn eventDAS-59122-7 produces an amplicon that is diagnostic for corn eventDAS-59122-7 ; (b) performing a nucleic acid amplification reaction,thereby producing the amplicon; and (c) detecting the amplicon.

DNA molecules that comprise the novel transgene/flanking insertionregion, SEQ ID NO: 21, 5′ flanking plus 1000 internal and SEQ ID NO: 22,3′ flanking plus 1000 internal and are homologous or complementary toSEQ ID NO: 21 and SEQ ID NO: 22 are an embodiment of this invention.

DNA sequences that comprise the novel transgene/flanking insertionregion, SEQ ID NO: 21 are an embodiment of this invention. DNA sequencesthat comprise a sufficient length of polynucleotides of transgene insertsequence and a sufficient length of polynucleotides of maize genomicand/or flanking sequence from maize plant DAS-59122-7 of SEQ ID NO: 21that are useful as primer sequences for the production of an ampliconproduct diagnostic for maize plant DAS-59122-7 are included.

In addition, DNA sequences that comprise the novel transgene/flankinginsertion region, SEQ ID NO: 22 are provided. DNA sequences thatcomprise a sufficient length of polynucleotides of transgene insertsequence and a sufficient length of polynucleotides of maize genomicand/or flanking sequence from maize plant DAS-59122-7 of SEQ ID NO: 22that are useful as primer sequences for the production of an ampliconproduct diagnostic for maize plant DAS-59122-7 are included.

According to another embodiment of the invention, the DNA sequences thatcomprise at least11 or more nucleotides of the transgene portion of theDNA sequence of SEQ ID NO: 21 or complements thereof, and a similarlength of 5′ flanking maize DNA sequence of SEQ ID NO: 21 or complementsthereof are useful as DNA primers in DNA amplification methods. Theamplicons produced using these primers are diagnostic for maize eventDAS-59122-7. Therefore, embodiments of the invention also include theamplicons produced by DNA primers homologous or complementary to SEQ IDNO: 21.

According to another embodiment of the invention, the DNA sequences thatcomprise at least11 or more nucleotides of the transgene portion of theDNA sequence of SEQ ID NO: 22 or complements thereof, and a similarlength of 3′ flanking maize DNA sequence of SEQ ID NO: 22 or complementsthereof are useful as DNA primers in DNA amplification methods. Theamplicons produced using these primers are diagnostic for maize eventDAS-59122-7. Therefore, embodiments of the invention also include theamplicons produced by DNA primers homologous or complementary to SEQ IDNO: 22.

More specifically, a pair of DNA molecules comprising a DNA primer set,wherein the DNA molecules are identified as SEQ ID NO: 18 or complementsthereof and SEQ ID NO: 1 or complements thereof; SEQ ID NO: 2 orcomplements thereof and SEQ ID NO: 17 or complements thereof; SEQ ID NO:10 or complements thereof and SEQ ID NO: 9 or complements thereof; SEQID NO: 8 or complements thereof and SEQ ID NO: 17 or complementsthereof; and SEQ ID NO: 36 or complements thereof and SEQ ID NO: 37 orcomplements thereof are embodiments of the invention.

Further embodiments of the invention include the amplicon comprising theDNA molecules of SEQ ID NO: 18 and SEQ ID NO: 1; the amplicon comprisingthe DNA molecules of SEQ ID NO: 2 and SEQ ID NO: 17; the ampliconcomprising the DNA molecules of SEQ ID NO: 10 and SEQ ID NO: 9; theamplicon comprising the DNA molecules of SEQ ID NO: 8 and SEQ ID NO: 17;and the amplicon comprising the DNA molecules of SEQ ID NO: 36 and SEQID NO: 37.

Further embodiments of the invention include the following primers,which are useful in detecting or characterizing event DAS-59122-7 : SEQID NO: 11 or complements thereof; SEQ ID NO: 5 or complements thereof;SEQ ID NO: 4 or complements thereof; SEQ ID NO: 7 or complementsthereof; SEQ ID NO: 6 or complements thereof; SEQ ID NO: 3 orcomplements thereof; SEQ ID NO: 18 or complements thereof; SEQ ID NO: 14or complements thereof; SEQ ID NO: 13 or complements thereof; SEQ ID NO:15 or complements thereof; SEQ ID NO: 17 or complements thereof; SEQ IDNO: 16 or complements thereof; and SEQ ID NO: 12 or complements thereof.Further embodiments also include the amplicons produced by pairing anyof the primers listed above.

According to another embodiment of the invention, methods of detectingthe presence of a DNA molecule corresponding to the DAS-59122-7 event ina sample, such methods comprising: (a) contacting the sample comprisingDNA extracted from a corn plant with a DNA probe, molecule thathybridizes under stringent hybridization conditions with DNA extractedfrom corn event DAS-59122-7 and does not hybridize under the stringenthybridization conditions with a control corn plant DNA; (b) subjectingthe sample and probe to stringent hybridization conditions; and (c)detecting hybridization of the probe to the DNA. More specifically, amethod for detecting the presence of a DNA molecule corresponding to theDAS-59122-7 event in a sample, such methods, consisting of (a)contacting the sample comprising DNA extracted from a corn plant with aDNA probe molecule that consists of sequences that are unique to theevent, e.g. junction sequences, wherein said DNA probe moleculehybridizes under stringent hybridization conditions with DNA extractedfrom corn event DAS-59122-7 and does not hybridize under the stringenthybridization conditions with a control corn plant DNA; (b) subjectingthe sample and probe to stringent hybridization conditions; and (c)detecting hybridization of the probe to the DNA.

In addition, a kit and methods for identifying event DAS-59122-7 in abiological sample which detects a DAS-59122-7 specific region within SEQID NO: 23 are provided.

DNA molecules are provided that comprise at least one junction sequenceof DAS-59122-7 selected from the group consisting of SEQ ID NO: 32, 33,34, and 35 and complements thereof; wherein a junction sequence spansthe junction between heterologous DNA inserted into the genome and theDNA from the corn cell flanking the insertion site, i.e. flanking DNA,and is diagnostic for the DAS-59122-7 event.

According to another embodiment of the invention, methods of producingan insect resistant corn plant that comprise the steps of: (a) sexuallycrossing a first parental corn line comprising the expression cassettesof the invention, which confers resistance to insects, and a secondparental corn line that lacks insect resistance, thereby producing aplurality of progeny plants; and (b) selecting a progeny plant that isinsect resistant. Such methods may optionally comprise the further stepof back-crossing the progeny plant to the second parental corn line toproducing a true-breeding corn plant that is insect resistant.

A further embodiment of the invention provides a method of producing acorn plant that is resistant to insects comprising transforming a corncell with the DNA construct PHI17662A (SEQ ID NO: 24), growing thetransformed corn cell into a corn plant, selecting the corn plant thatshows resistance to insects, and further growing the corn plant into afertile corn plant. The fertile corn plant can be self pollinated orcrossed with compatible corn varieties to produce insect resistantprogeny.

Another embodiment of the invention further relates to a DNA detectionkit for identifying maize event DAS-59122-7 in biological samples. Thekit comprises a first primer which specifically recognizes the 5′ or 3′flanking region of DAS-59122-7, and a second primer which specificallyrecognizes a sequence within the foreign DNA of DAS-59122-7, or withinthe flanking DNA, for use in a PCR identification protocol. A furtherembodiment of the invention relates to a kit for identifying eventDAS-59122-7 in biological samples, which kit comprises a specific probehaving a sequence which corresponds or is complementary to, a sequencehaving between 80% and 100% sequence identity with a specific region ofevent DAS-59122-7. The sequence of the probe corresponds to a specificregion comprising part of the 5′ or 3′ flanking region of eventDAS-59122-7.

The methods and kits encompassed by the embodiments of the presentinvention can be used for different purposes such as, but not limited tothe following: to identify event DAS-69122-7 in plants, plant materialor in products such as, but not limited to, food or feed products (freshor processed) comprising, or derived from plant material; additionallyor alternatively, the methods and kits can be used to identifytransgenic plant material for purposes of segregation between transgenicand non-transgenic material; additionally or alternatively, the methodsand kits can be used to determine the quality of plant materialcomprising maize event DAS-59122-7. The kits may also contain thereagents and materials necessary for the performance of the detectionmethod.

A further embodiment of this invention relates to the DAS-59122-7 cornplant or its parts, including, but not limited to, pollen, ovules,vegetative cells, the nuclei of pollen cells, and the nuclei of eggcells of the corn plant DAS-59122-7 and the progeny derived thereof. Thecorn plant and seed DAS-59122-7 from which the DNA primer moleculesprovide a specific amplicon product is an embodiment of the invention.

The foregoing and other aspects of the invention will become moreapparent from the following detailed description and accompanyingdrawing.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. DNA sequence (SEQ ID NO: 23) showing the transgenic insertPHI17662A, as well as the sequences flanking the transgenic insert. The5′ and 3′ border regions, bp 1 to bp 2593 and bp 9937 to bp 11922,respectively, are underlined. Two nucleotide differences (bp 6526 and bp6562) based on comparison to the transforming plasmid PHP17662 are notedin bold and underlined.

FIG. 2. Schematic diagram of the B.t. Cry34/35Ab1 event DAS-59122-7insert region is divided into three separate sections; the 5′ borderregion with corn genomic DNA, the intact T-DNA insert, and the 3′ borderregion with corn genomic DNA. The two arrows beneath the diagram of theinsert indicate the start and end points of the sequence derived from 5′and 3′ genome walking fragments. Other boxes beneath the diagram of theinsert represent PCR fragments that were amplified from genomic DNA ofevent DAS-59122-7 and sequenced to cover the intact T-DNA insert and the5′ and 3′ insert/border junction regions.

FIG. 3. Schematic diagram of the B.t. Cry34/35Ab1 event DAS-59122-7insert region is divided into three separate sections; the 5′ borderregion with corn genomic DNA, the intact T-DNA insert, and the 3′ borderregion with corn genomic DNA. Boxes beneath the diagram of the insertrepresent PCR fragments located in either the genomic border regions oracross the 5′ and 3′ junction regions of the T-DNA insert with corngenomic DNA that were amplified from genomic DNA from event DAS-59122-7.

DETAILED DESCRIPTION

The following definitions and methods are provided to better define thepresent invention and to guide those of ordinary skill in the art in thepractice of the present invention. Unless otherwise noted, terms are tobe understood according to conventional usage by those of ordinary skillin the relevant art. Definitions of common terms in molecular biologymay also be found in Rieger et al., Glossary of Genetics: Classical andMolecular, 5^(th) edition, Springer-Verlag; New York, 1991; and Lewin,Genes V, Oxford University Press: New York, 1994. The nomenclature forDNA bases as set forth at 37 CFR §1.822 is used.

As used herein, the term “comprising” means “including but not limitedto”.

As used herein, the term “corn” means Zea mays or maize and includes allplant varieties that can be bred with corn, including wild maizespecies.

As used herein, the term “DAS-59122-7 specific” refers to a nucleotidesequence which is suitable for discriminatively identifying eventDAS-59122-7 in plants, plant material, or in products such as, but notlimited to, food or feed products (fresh or processed) comprising, orderived from plant material.

As used herein, the terms “insect resistant” and “impacting insectpests” refers to effecting changes in insect feeding, growth, and/orbehavior at any stage of development, including but not limited to:killing the insect; retarding growth; preventing reproductivecapability; inhibiting feeding; and the like.

As used herein, the terms “pesticidal activity” and “insecticidalactivity” are used synonymously to refer to activity of an organism or asubstance (such as, for example, a protein) that can be measured bynumerous parameters including, but not limited to, pest mortality, pestweight loss, pest attraction, pest repellency, and other behavioral andphysical changes of a pest after feeding on and/or exposure to theorganism or substance for an appropriate length of time. For example“pesticidal proteins” are proteins that display pesticidal activity bythemselves or in combination with other proteins.

“Coding sequence” refers to a nucleotide sequence that codes for aspecific amino acid sequence. As used herein, the terms “encoding” or“encoded” when used in the context of a specified nucleic acid mean thatthe nucleic acid comprises the requisite information to guidetranslation of the nucleotide sequence into a specified protein. Theinformation by which a protein is encoded is specified by the use ofcodons. A nucleic acid encoding a protein may comprise non-translatedsequences (e.g., introns) within translated regions of the nucleic acidor may lack such intervening non-translated sequences (e.g., as incDNA).

“Gene” refers to a nucleic acid fragment that expresses a specificprotein, including regulatory sequences preceding (5′ non-codingsequences) and following (3′ non-coding sequences) the coding sequence.“Native gene” refers to a gene as found in nature with its ownregulatory sequences. “Chimeric gene” refers any gene that is not anative gene, comprising regulatory and coding sequences that are notfound together in nature. Accordingly, a chimeric gene may compriseregulatory sequences and coding sequences that are derived fromdifferent sources, or regulatory sequences and coding sequences derivedfrom the same source, but arranged in a manner different than that foundin nature. “Endogenous gene” refers to a native gene in its naturallocation in the genome of an organism. “Foreign” refers to material notnormally found in the location of interest. Thus “foreign DNA” maycomprise both recombinant DNA as well as newly introduced, rearrangedDNA of the plant. A “foreign” gene refers to a gene not normally foundin the host organism, but that is introduced into the host organism bygene transfer. Foreign genes can comprise native genes inserted into anon-native organism, or chimeric genes. A “transgene” is a gene that hasbeen introduced into the genome by a transformation procedure. The sitein the plant genome where a recombinant DNA has been inserted may bereferred to as the “insertion site” or “target site”.

As used herein, “insert DNA” refers to the heterologous DNA within theexpression cassettes used to transform the plant material while“flanking DNA” can exist of either genomic DNA naturally present in anorganism such as a plant, or foreign (heterologous) DNA introduced viathe transformation process which is extraneous to the original insertDNA molecule, e.g. fragments associated with the transformation event. A“flanking region” or “flanking sequence” as used herein refers to asequence of at least twenty (20) base pair, preferably at least fifty(50) base pair, and up to five thousand (5000) base pair which islocated either immediately upstream of and contiguous with orimmediately downstream of and contiguous with the original foreigninsert DNA molecule. Transformation procedures leading to randomintegration of the foreign DNA will result in transformants containingdifferent flanking regions characteristic and unique for eachtransformant. When recombinant DNA is introduced into a plant throughtraditional crossing, its flanking regions will generally not bechanged. Transformants will also contain unique junctions between apiece of heterologous insert DNA and genomic DNA, or two (2) pieces ofgenomic DNA, or two (2) pieces of heterologous DNA. A “junction” is apoint where two (2) specific DNA fragments join. For example, a junctionexists where insert DNA joins flanking DNA. A junction point also existsin a transformed organism where two (2) DNA fragments join together in amanner that is modified from that found in the native organism.“Junction DNA” refers to DNA that comprises a junction point.

As used herein, “heterologous” in reference to a nucleic acid is anucleic acid that originates from a foreign species, or, if from thesame species, is substantially modified from its native form incomposition and/or genomic locus by deliberate human intervention. Forexample, a promoter operably linked to a heterologous nucleotidesequence can be from a species different from that from which thenucleotide sequence was derived, or, if from the same species, thepromoter is not naturally found operably linked to the nucleotidesequence. A heterologous protein may originate from a foreign species,or, if from the same species, is substantially modified from itsoriginal form by deliberate human intervention.

“Regulatory sequences” refer to nucleotide sequences located upstream(5′ non-coding sequences), within, or downstream (3′ non-codingsequences) of a coding sequence, and which influence the transcription,RNA processing or stability, or translation of the associated codingsequence. Regulatory sequences may include promoters, translation leadersequences, introns, and polyadenylation recognition sequences.

“Promoter” refers to a nucleotide sequence capable of controlling theexpression of a coding sequence or functional RNA. In general, a codingsequence is located 3′ to a promoter sequence. The promoter sequenceconsists of proximal and more distal upstream elements, the latterelements are often referred to as enhancers. Accordingly, an “enhancer”is a nucleotide sequence that can stimulate promoter activity and may bean innate element of the promoter or a heterologous element inserted toenhance the level or tissue-specificity of a promoter. Promoters may bederived in their entirety from a native gene, or be composed ofdifferent elements derived from different promoters found in nature, oreven comprise synthetic nucleotide segments. It is understood by thoseskilled in the art that different promoters may direct the expression ofa gene in different tissues or cell types, or at different stages ofdevelopment, or in response to different environmental conditions.Promoters that cause a nucleic acid fragment to be expressed in mostcell types at most times are commonly referred to as “constitutivepromoters”. New promoters of various types useful in plant cells areconstantly being discovered; numerous examples may be found in thecompilation by Okamuro and Goldberg (1989) Biochemistry of Plants15:1-82. It is further recognized that since in most cases the exactboundaries of regulatory sequences have not been completely defined,nucleic acid fragments of different lengths may have identical promoteractivity.

The “translation leader sequence” refers to a nucleotide sequencelocated between the promoter sequence of a gene and the coding sequence.The translation leader sequence is present in the fully processed mRNAupstream of the translation start sequence. The translation leadersequence may affect numerous parameters including, processing of theprimary transcript to mRNA, mRNA stability and/or translationefficiency. Examples of translation leader sequences have been described(Turner and Foster (1995) Mol. Biotechnol. 3:225-236).

The “3′ non-coding sequences” refer to nucleotide sequences locateddownstream of a coding sequence and include polyadenylation recognitionsequences and other sequences encoding regulatory signals capable ofaffecting mRNA processing or gene expression. The polyadenylation signalis usually characterized by affecting the addition of polyadenylic acidtracts to the 3′ end of the mRNA precursor. The use of different 3′non-coding sequences is exemplified by Ingelbrecht et al. (1989) PlantCell 1:671-680.

A “protein” or “polypeptide” is a chain of amino acids arranged in aspecific order determined by the coding sequence in a polynucleotideencoding the polypeptide.

A DNA construct is an assembly of DNA molecules linked together thatprovide one or more expression cassettes. The DNA construct may be aplasmid that is enabled for self replication in a bacterial cell andcontains various endonuclease enzyme restriction sites that are usefulfor introducing DNA molecules that provide functional genetic elements,i.e., promoters, introns, leaders, coding sequences, 3′ terminationregions, among others; or a DNA construct may be a linear assembly ofDNA molecules, such as an expression cassette. The expression cassettecontained within a DNA construct comprise the necessary genetic elementsto provide transcription of a messenger RNA. The expression cassette canbe designed to express in prokaryote cells or eukaryotic cells.Expression cassettes of the embidoments of the present invention aredesigned to express in plant cells.

The DNA molecules of embodiments of the invention are provided inexpression cassettes for expression in an organism of interest. Thecassette will include 5′ and 3′ regulatory sequences operably linked toa coding sequence. “Operably linked” means that the nucleic acidsequences being linked are contiguous and, where necessary to join twoprotein coding regions, contiguous and in the same reading frame.Operably linked is intended to indicate a functional linkage between apromoter and a second sequence, wherein the promoter sequence initiatesand mediates transcription of the DNA sequence corresponding to thesecond sequence. The cassette may additionally contain at least oneadditional gene to be cotransformed into the organism. Alternatively,the additional gene(s) can be provided on multiple expression cassettesor multiple DNA constructs.

The expression cassette will include in the 5′ to 3′ direction oftranscription: a transcriptional and translational initiation region, acoding region, and a transcriptional and translational terminationregion functional in the organism serving as a host. The transcriptionalinitiation region (i.e., the promoter) may be native or analogous, orforeign or heterologous to the host organism. Additionally, the promotermay be the natural sequence or alternatively a synthetic sequence. Theexpression cassettes may additionally contain 5′ leader sequences in theexpression cassette construct. Such leader sequences can act to enhancetranslation.

It is to be understood that as used herein the term “transgenic”includes any cell, cell line, callus, tissue, plant part, or plant, thegenotype of which has been altered by the presence of a heterologousnucleic acid including those transgenics initially so altered as well asthose created by sexual crosses or asexual propagation from the initialtransgenic. The term “transgenic” as used herein does not encompass thealteration of the genome (chromosomal or extra-chromosomal) byconventional plant breeding methods or by naturally occurring eventssuch as random cross-fertilization, non-recombinant viral infection,non-recombinant bacterial transformation, non-recombinant transposition,or spontaneous mutation.

A transgenic “event” is produced by transformation of plant cells with aheterologous DNA construct(s), including a nucleic acid expressioncassette that comprises a transgene of interest, the regeneration of apopulation of plants resulting from the insertion of the transgene intothe genome of the plant, and selection of a particular plantcharacterized by insertion into a particular genome location. An eventis characterized phenotypically by the expression of the transgene. Atthe genetic level, an event is part of the genetic makeup of a plant.The term “event” also refers to progeny produced by a sexual outcrossbetween the transformant and another variety that include theheterologous DNA. Even after repeated back-crossing to a recurrentparent, the inserted DNA and flanking DNA from the transformed parent ispresent in the progeny of the cross at the same chromosomal location.The term “event” also refers to DNA from the original transformantcomprising the inserted DNA and flanking sequence immediately adjacentto the inserted DNA that would be expected to be transferred to aprogeny that receives inserted DNA including the transgene of interestas the result of a sexual cross of one parental line that includes theinserted DNA (e.g., the original transformant and progeny resulting fromselfing) and a parental line that does not contain the inserted DNA.

An insect resistant DAS-59122-7 corn plant can be bred by first sexuallycrossing a first parental corn plant consisting of a corn plant grownfrom the transgenic DAS-59122-7 corn plant and progeny thereof derivedfrom transformation with the expression cassettes of the embodiments ofthe present invention that confers insect resistance, and a secondparental corn plant that lacks insect resistance, thereby producing aplurality of first progeny plants; and then selecting a first progenyplant that is resistant to insects; and selling the first progeny plant,thereby producing a plurality of second progeny plants; and thenselecting from the second progeny plants an insect resistant plant.These steps can further include the back-crossing of the first insectresistant progeny plant or the second insect resistant progeny plant tothe second parental corn plant or a third parental corn plant, therebyproducing a corn plant that is resistant to insects.

As used herein, the term “plant” includes reference to whole plants,plant organs (e.g., leaves, stems, roots, etc.), seeds, plant cells, andprogeny of same. Parts of transgenic plants understood to be within thescope of the invention comprise, for example, plant cells, protoplasts,tissues, callus, embryos as well as flowers, stems, fruits, leaves, androots originating in transgenic plants or their progeny previouslytransformed with a DNA molecule of the invention and thereforeconsisting at least in part of transgenic cells, are also an embodimentof the present invention.

As used herein, the term “plant cell” includes, without limitation,seeds, suspension cultures, embryos, meristematic regions, callustissue, leaves, roots, shoots, gametophytes, sporophytes, pollen, andmicrospores. The class of plants that can be used in the methods of theinvention is generally as broad as the class of higher plants amenableto transformation techniques, including both monocotyledonous anddicotyledonous plants.

“Transformation” refers to the transfer of a nucleic acid fragment intothe genome of a host organism, resulting in genetically stableinheritance. Host organisms containing the transformed nucleic acidfragments are referred to as “transgenic” organisms. Examples of methodsof plant transformation include Agrobacterium-mediated transformation(De Blaere et al. (1987) Meth. Enzymol. 143:277) andparticle-accelerated or “gene gun” transformation technology (Klein etal. (1987) Nature (London) 327:70-73; U.S. Pat. No. 4,945,050,incorporated herein by reference). Additional transformation methods aredisclosed below.

Thus, isolated polynucleotides of the invention can be incorporated intorecombinant constructs, typically DNA constructs, which are capable ofintroduction into and replication in a host cell. Such a construct canbe a vector that includes a replication system and sequences that arecapable of transcription and translation of a polypeptide-encodingsequence in a given host cell. A number of vectors suitable for stabletransfection of plant cells or for the establishment of transgenicplants have been described in, e.g., Pouwels et al., (1985; Supp. 1987)Cloning Vectors: A Laboratory Manual, Weissbach and Weissbach (1989)Methods for Plant Molecular Biology, (Academic Press, New York); andFlevin et al., (1990) Plant Molecular Biology Manual, (Kluwer AcademicPublishers). Typically, plant expression vectors include, for example,one or more cloned plant genes under the transcriptional control of 5′and 3′ regulatory sequences and a dominant selectable marker. Such plantexpression vectors also can contain a promoter regulatory region (e.g.,a regulatory region controlling inducible or constitutive,environmentally- or developmentally-regulated, or cell- ortissue-specific expression), a transcription initiation start site, aribosome binding site, an RNA processing signal, a transcriptiontermination site, and/or a polyadenylation signal.

It is also to be understood that two different transgenic plants canalso be mated to produce offspring that contain two independentlysegregating added, exogenous genes. Selfing of appropriate progeny canproduce plants that are homozygous for both added, exogenous genes.Back-crossing to a parental plant and out-crossing with a non-transgenicplant are also contemplated, as is vegetative propagation. Descriptionsof other breeding methods that are commonly used for different traitsand crops can be found in one of several references, e.g., Fehr, inBreeding Methods for Cultivar Development, Wilcos J. ed., AmericanSociety of Agronomy, Madison Wis. (1987).

A “probe” is an isolated nucleic acid to which is attached aconventional detectable label or reporter molecule, e.g., a radioactiveisotope, ligand, chemiluminescent agent, or enzyme. Such a probe iscomplementary to a strand of a target nucleic acid, in the case of thepresent invention, to a strand of isolated DNA from corn eventDAS-59122-7 whether from a corn plant or from a sample that includes DNAfrom the event. Probes according to the present invention include notonly deoxyribonucleic or ribonucleic acids but also polyamides and otherprobe materials that bind specifically to a target DNA sequence and canbe used to detect the presence of that target DNA sequence.

“Primers” are isolated nucleic acids that are annealed to acomplementary target DNA strand by nucleic acid hybridization to form ahybrid between the primer and the target DNA strand, then extended alongthe target DNA strand by a polymerase, e.g., a DNA polymerase. Primerpairs of the invention refer to their use for amplification of a targetnucleic acid sequence, e.g., by the polymerase chain reaction (PCR) orother conventional nucleic-acid amplification methods. “PCR” or“polymerase chain reaction” is a technique used for the amplification ofspecific DNA segments (see, U.S. Pat. Nos. 4,683,195 and 4,800,159;herein incorporated by reference).

Probes and primers are of sufficient nucleotide length to bind to thetarget DNA sequence specifically in the hybridization conditions orreaction conditions determined by the operator. This length may be ofany length that is of sufficient length to be useful in a detectionmethod of choice. Generally, eleven (11) nucleotides or more in length,eighteen (18) nucleotides or more, and twenty-two (22) nucleotides ormore, are used. Such probes and primers hybridize specifically to atarget sequence under high stringency hybridization conditions. Probesand primers according to embodiments of the present invention may havecomplete DNA sequence similarity of contiguous nucleotides with thetarget sequence, although probes differing from the target DNA sequenceand that retain the ability to hybridize to target DNA sequences may bedesigned by conventional methods. Probes can be used as primers, but aregenerally designed to bind to the target DNA or RNA and are not used inan amplification process.

Specific primers can be used to amplify an integration fragment toproduce an amplicon that can be used as a “specific probe” foridentifying event DAS-59122-7 in biological samples. When the probe ishybridized with the nucleic acids of a biological sample underconditions which allow for the binding of the probe to the sample, thisbinding can be detected and thus allow for an indication of the presenceof event DAS-59122-7 in the biological sample. Such identification of abound probe has been described in the art. In an embodiment of theinvention the specific probe is a sequence which, under optimizedconditions, hybridizes specifically to a region within the 5′ or 3′flanking region of the event and also comprises a part of the foreignDNA contiguous therewith. The specific probe may comprise a sequence ofat least 80%, between 80 and 85%, between 85 and 90%, between 90 and95%, and between 95 and 100% identical (or complementary) to a specificregion of the event.

Methods for preparing and using probes and primers are described, forexample, in Molecular Cloning: A Laboratory Manual, 2^(nd) ed., vol.1-3,ed. Sambrook et al, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. 1989 (hereinafter, “Sambrook et al, 1989”); CurrentProtocols in Molecular Biology, ed. Ausubel et al, Greene Publishing andWiley-Interscience, New York, 1992 (with periodic updates) (hereinafter,“Ausubel et al., 1992”); and Innis et al., PCR Protocols: A Guide toMethods and Applications, Academic Press: San Diego, 1990. PCR primerpairs can be derived from a known sequence, for example, by usingcomputer programs intended for that purpose such as the PCR primeranalysis tool in Vector NTI version 6 (Informax Inc., Bethesda Md.);PrimerSelect (DNASTAR Inc., Madison, Wis.); and Primer (Version 0.5©,1991, Whitehead Institute for Biomedical Research, Cambridge, Mass.).Additionally, the sequence can be visually scanned and primers manuallyidentified using guidelines known to one of skill in the art.

A “kit” as used herein refers to a set of reagents for the purpose ofperforming the method embodiments of the invention, more particularly,the identification of the event DAS-59122-7 in biological samples. Thekit of the invention can be used, and its components can be specificallyadjusted, for purposes of quality control (e.g. purity of seed lots),detection of event DAS-59122-7 in plant material, or material comprisingor derived from plant material, such as but not limited to food or feedproducts. “Plant material” as used herein refers to material which isobtained or derived from a plant.

Primers and probes based on the flanking DNA and insert sequencesdisclosed herein can be used to confirm (and, if necessary, to correct)the disclosed sequences by conventional methods, e.g., by re-cloning andsequencing such sequences. The nucleic acid probes and primers of thepresent invention hybridize under stringent conditions to a target DNAsequence. Any conventional nucleic acid hybridization or amplificationmethod can be used to identify the presence of DNA from a transgenicevent in a sample. Nucleic acid molecules or fragments thereof arecapable of specifically hybridizing to other nucleic acid moleculesunder certain circumstances. As used herein, two nucleic acid moleculesare said to be capable of specifically hybridizing to one another if thetwo molecules are capable of forming an anti-parallel, double-strandednucleic acid structure.

A nucleic acid molecule is said to be the “complement” of anothernucleic acid molecule if they exhibit complete complementarity. As usedherein, molecules are said to exhibit “complete complementarity” whenevery nucleotide of one of the molecules is complementary to anucleotide of the other. Two molecules are said to be “minimallycomplementary” if they can hybridize to one another with sufficientstability to permit them to remain annealed to one another under atleast conventional “low-stringency” conditions. Similarly, the moleculesare said to be “complementary” if they can hybridize to one another withsufficient stability to permit them to remain annealed to one anotherunder conventional “high-stringency” conditions. Conventional stringencyconditions are described by Sambrook et al., 1989, and by Haymes et al.,In: Nucleic Acid Hybridization, a Practical Approach, IRL Press,Washington, D.C. (1985), departures from complete complementarity aretherefore permissible, as long as such departures do not completelypreclude the capacity of the molecules to form a double-strandedstructure. In order for a nucleic acid molecule to serve as a primer orprobe it need only be sufficiently complementary in sequence to be ableto form a stable double-stranded structure under the particular solventand salt concentrations employed.

In hybridization reactions, specificity is typically the function ofpost-hybridization washes, the critical factors being the ionic strengthand temperature of the final wash solution. The thermal melting point(Tm) is the temperature (under defined ionic strength and pH) at which50% of a complementary target sequence hybridizes to a perfectly matchedprobe. For DNA-DNA hybrids, the Tm can be approximated from the equationof Meinkoth and Wahl (1984) Anal. Biochem. 138:267-284: Tm=81.5° C.+16.6(log M)+0.41 (% GC)−0.61 (% form)−500/L; where M is the molarity ofmonovalent cations, % GC is the percentage of guanosine and cytosinenucleotides in the DNA, % form is the percentage of formamide in thehybridization solution, and L is the length of the hybrid in base pairs.Tm is reduced by about 1° C. for each 1% of mismatching; thus, Tm,hybridization, and/or wash conditions can be adjusted to hybridize tosequences of the desired identity. For example, if sequences with >90%identity are sought, the Tm can be decreased 10° C. Generally, stringentconditions are selected to be about 5° C. lower than the Tm for thespecific sequence and its complement at a defined ionic strength and pH.However, severely stringent conditions can utilize a hybridizationand/or wash at 1, 2, 3, or 4° C. lower than the Tm; moderately stringentconditions can utilize a hybridization and/or wash at 6, 7, 8, 9, or 10°C. lower than the Tm; low stringency conditions can utilize ahybridization and/or wash at 11, 12, 13, 14, 15, or 20° C. lower thanthe Tm.

Using the equation, hybridization and wash compositions, and desired Tm,those of ordinary skill will understand that variations in thestringency of hybridization and/or wash solutions are inherentlydescribed. If the desired degree of mismatching results in a Tm of lessthan 45° C. (aqueous solution) or 32° C. (formamide solution), it ispreferred to increase the SSC concentration so that a higher temperaturecan be used. An extensive guide to the hybridization of nucleic acids isfound in Tijssen (1993) Laboratory Techniques in Biochemistry andMolecular Biology—Hybridization with Nucleic Acid Probes, Part I,Chapter 2 (Elsevier, N.Y.); and Ausubel et al., eds. (1995) CurrentProtocols in Molecular Biology, Chapter 2 (Greene Publishing andWiley-Interscience, New York). See Sambrook et al. (1989) MolecularCloning: A Laboratory Manual (2d ed., Cold Spring Harbor LaboratoryPress, Plainview, N.Y.).

As used herein, a substantially homologous sequence is a nucleic acidmolecule that will specifically hybridize to the complement of thenucleic acid molecule to which it is being compared under highstringency conditions. Appropriate stringency conditions which promoteDNA hybridization, for example, 6×sodium chloride/sodium citrate (SSC)at about 45° C., followed by a wash of 2×SSC at 50° C., are known tothose skilled in the art or can be found in Current Protocols inMolecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6.Typically, stringent conditions will be those in which the saltconcentration is less than about 1.5 M Na ion, typically about 0.01 to1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and thetemperature is at least about 30° C. for short probes (e.g., 10 to 50nucleotides) and at least about 60° C. for long probes (e.g., greaterthan 50 nucleotides). Stringent conditions may also be achieved with theaddition of a destabilizing agent such as formamide. Exemplary lowstringency conditions include hybridization with a buffer solution of 30to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulphate) at 37° C.,and a wash in 1× to 2×SSC (20×SSC=3.0 M NaCl/0.3 M trisodium citrate) at50 to 55° C. Exemplary moderate stringency conditions includehybridization in 40 to 45% formamide, 1 M NaCl, 1% SDS at 37° C., and awash in 0.5× to 1×SSC at 55 to 60° C. Exemplary high stringencyconditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at37° C., and a wash in 0.1×SSC at 60 to 65° C. A nucleic acid of theinvention may specifically hybridize to one or more of the nucleic acidmolecules unique to the DAS-59122-7 event or complements thereof orfragments of either under moderately stringent conditions.

Methods of alignment of sequences for comparison are well known in theart. Thus, the determination of percent identity between any twosequences can be accomplished using a mathematical algorithm.Non-limiting examples of such mathematical algorithms are the algorithmof Myers and Miller (1988) CABIOS 4:11-17; the local homology algorithmof Smith et al. (1981) Adv. Appl. Math. 2:482; the homology alignmentalgorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443-453; thesearch-for-similarity-method of Pearson and Lipman (1988) Proc. Natl.Acad. Sci. 85:2444-2448; the algorithm of Karlin and Altschul (1990)Proc. Natl. Acad. Sci. USA 87:2264, modified as in Karlin and Altschul(1993) Proc. Natl. Acad. Sci. USA 90:5873-5877.

Computer implementations of these mathematical algorithms can beutilized for comparison of sequences to determine sequence identity.Such implementations include, but are not limited to: CLUSTAL in thePC/Gene program (available from Intelligenetics, Mountain View, Calif.);the ALIGN program (Version 2.0); the ALIGN PLUS program (version 3.0,copyright 1997); and GAP, BESTFIT, BLAST, FASTA, and TFASTA in theWisconsin Genetics Software Package, Version 10 (available fromAccelrys, 9685 Scranton Road, San Diego, Calif. 92121, USA). Alignmentsusing these programs can be performed using the default parameters.

The CLUSTAL program is well described by Higgins and Sharp, Gene 73:237-244 (1988); Higgins and Sharp, CABIOS 5: 151-153 (1989); Corpet, etal., Nucleic Acids Research 16: 10881-90 (1988); Huang, et al., ComputerApplications in the Biosciences 8: 155-65 (1992), and Pearson, et al.,Methods in Molecular Biology 24: 307-331 (1994). The ALIGN and the ALIGNPLUS programs are based on the algorithm of Myers and Miller (1988)supra. The BLAST programs of Altschul et al. (1990) J. Mol. Biol.215:403 are based on the algorithm of Karlin and Altschul (1990) supra.The BLAST family of programs which can be used for database similaritysearches includes: BLASTN for nucleotide query sequences againstnucleotide database sequences; BLASTX for nucleotide query sequencesagainst protein database sequences; BLASTP for protein query sequencesagainst protein database sequences; TBLASTN for protein query sequencesagainst nucleotide database sequences; and TBLASTX for nucleotide querysequences against nucleotide database sequences. See, Current Protocolsin Molecular Biology, Chapter 19, Ausubel, et al., Eds., GreenePublishing and Wiley-Interscience, New York (1995). Alignment may alsobe performed manually by visual inspection.

To obtain gapped alignments for comparison purposes, Gapped BLAST (inBLAST 2.0) can be utilized as described in Altschul et al. (1997)Nucleic Acids Res. 25:3389. Alternatively, PSI-BLAST (in BLAST 2.0) canbe used to perform an iterated search that detects distant relationshipsbetween molecules. See Altschul et al. (1997) supra. When utilizingBLAST, Gapped BLAST, PSI-BLAST, the default parameters of the respectiveprograms (e.g., BLASTN for nucleotide sequences, BLASTX for proteins)can be used. See www.ncbi.hlm.nih.gov.

As used herein, “sequence identity” or “identity” in the context of twonucleic acid or polypeptide sequences makes reference to the residues inthe two sequences that are the same when aligned for maximumcorrespondence over a specified comparison window. When percentage ofsequence identity is used in reference to proteins it is recognized thatresidue positions which are not identical often differ by conservativeamino acid substitutions, where amino acid residues are substituted forother amino acid residues with similar chemical properties (e.g., chargeor hydrophobicity) and therefore do not change the functional propertiesof the molecule. When sequences differ in conservative substitutions,the percent sequence identity may be adjusted upwards to correct for theconservative nature of the substitution. Sequences that differ by suchconservative substitutions are said to have “sequence similarity” or“similarity”. Means for making this adjustment are well known to thoseof skill in the art. Typically this involves scoring a conservativesubstitution as a partial rather than a full mismatch, therebyincreasing the percentage sequence identity. Thus, for example, where anidentical amino acid is given a score of 1 and a non-conservativesubstitution is given a score of zero, a conservative substitution isgiven a score between zero and 1. The scoring of conservativesubstitutions is calculated, e.g., as implemented in the program PC/GENE(Intelligenetics, Mountain View, Calif.).

As used herein, “percentage of sequence identity” means the valuedetermined by comparing two optimally aligned sequences over acomparison window, wherein the portion of the polynucleotide sequence inthe comparison window may comprise additions or deletions (i.e., gaps)as compared to the reference sequence (which does not comprise additionsor deletions) for optimal alignment of the two sequences. The percentageis calculated by determining the number of positions at which theidentical nucleic acid base or amino acid residue occurs in bothsequences to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the window ofcomparison, and multiplying the result by 100 to yield the percentage ofsequence identity.

Regarding the amplification of a target nucleic acid sequence (e.g., byPCR) using a particular amplification primer pair, “stringentconditions” are conditions that permit the primer pair to hybridize onlyto the target nucleic-acid sequence to which a primer having thecorresponding wild-type sequence (or its complement) would bind andpreferably to produce a unique amplification product, the amplicon, in aDNA thermal amplification reaction.

The term “specific for (a target sequence)” indicates that a probe orprimer hybridizes under stringent hybridization conditions only to thetarget sequence in a sample comprising the target sequence.

As used herein, “amplified DNA” or “amplicon” refers to the product ofnucleic acid amplification of a target nucleic acid sequence that ispart of a nucleic acid template. For example, to determine whether acorn plant resulting from a sexual cross contains transgenic eventgenomic DNA from the corn plant of the invention, DNA extracted from thecorn plant tissue sample may be subjected to a nucleic acidamplification method using a DNA primer pair that includes a firstprimer derived from flanking sequence adjacent to the insertion site ofinserted heterologous DNA, and a second primer derived from the insertedheterologous DNA to produce an amplicon that is diagnostic for thepresence of the event DNA. Alternatively, the second primer may bederived from the flanking sequence. The amplicon is of a length and hasa sequence that is also diagnostic for the event. The amplicon may rangein length from the combined length of the primer pairs plus onenucleotide base pair to any length of amplicon producible by a DNAamplification protocol. Alternatively, primer pairs can be derived fromflanking sequence on both sides of the inserted DNA so as to produce anamplicon that includes the entire insert nucleotide sequence of thePHI17662A expression construct as well as the sequence flanking thetransgenic insert, see FIG. 1 (SEQ ID NO: 23), approximately twelve (12)Kb in size. A member of a primer pair derived from the flanking sequencemay be located a distance from the inserted DNA sequence, this distancecan range from one nucleotide base pair up to the limits of theamplification reaction, or about twenty thousand nucleotide base pairs.The use of the term “amplicon” specifically excludes primer dimers thatmay be formed in the DNA thermal amplification reaction.

Nucleic acid amplification can be accomplished by any of the variousnucleic acid amplification methods known in the art, including thepolymerase chain reaction (PCR). A variety of amplification methods areknown in the art and are described, inter alia, in U.S. Pat. Nos.4,683,195 and 4,683,202 and in PCR Protocols: A Guide to Methods andApplications, ed. Innis et al., Academic press, San Diego, 1990. PCRamplification methods have been developed to amplify up to 22 Kb ofgenomic DNA and up to 42 Kb of bacteriophage DNA (Cheng et al., Proc.Natl. Acad. Sci. USA 91:5695-5699, 1994). These methods as well as othermethods known in the art of DNA amplification may be used in thepractice of the embodiments of the present invention. It is understoodthat a number of parameters in a specific PCR protocol may need to beadjusted to specific laboratory conditions and may be slightly modifiedand yet allow for the collection of similar results. These adjustmentswill be apparent to a person skilled in the art.

The amplicon produced by these methods may be detected by a plurality oftechniques, including, but not limited to, Genetic Bit Analysis(Nikiforov, et al. Nucleic Acid Res. 22:4167-4175, 1994) where a DNAoligonucleotide is designed which overlaps both the adjacent flankingDNA sequence and the inserted DNA sequence. The oligonucleotide isimmobilized in wells of a microwell plate. Following PCR of the regionof interest (using one primer in the inserted sequence and one in theadjacent flanking sequence) a single-stranded PCR product can behybridized to the immobilized oligonucleotide and serve as a templatefor a single base extension reaction using a DNA polymerase and labeledddNTPs specific for the expected next base. Readout may be fluorescentor ELISA-based. A signal indicates presence of the insert/flankingsequence due to successful amplification, hybridization, and single baseextension.

Another detection method is the Pyrosequencing technique as described byWinge (Innov. Pharma. Tech. 00: 18-24, 2000). In this method anoligonucleotide is designed that overlaps the adjacent DNA and insertDNA junction. The oligonucleotide is hybridized to a single-stranded PCRproduct from the region of interest (one primer in the inserted sequenceand one in the flanking sequence) and incubated in the presence of a DNApolymerase, ATP, sulfurylase, luciferase, apyrase, adenosine 5′phosphosulfate and luciferin. dNTPs are added individually and theincorporation results in a light signal which is measured. A lightsignal indicates the presence of the transgene insert/flanking sequencedue to successful amplification, hybridization, and single or multi-baseextension.

Fluorescence Polarization as described by Chen et al., (Genome Res.9:492-498, 1999) is also a method that can be used to detect an ampliconof the invention. Using this method an oligonucleotide is designed whichoverlaps the flanking and inserted DNA junction. The oligonucleotide ishybridized to a single-stranded PCR product from the region of interest(one primer in the inserted DNA and one in the flanking DNA sequence)and incubated in the presence of a DNA polymerase and afluorescent-labeled ddNTP. Single base extension results inincorporation of the ddNTP. Incorporation can be measured as a change inpolarization using a fluorometer. A change in polarization indicates thepresence of the transgene insert/flanking sequence due to successfulamplification, hybridization, and single base extension.

Taqman® (PE Applied Biosystems, Foster City, Calif.) is described as amethod of detecting and quantifying the presence of a DNA sequence andis fully understood in the instructions provided by the manufacturer.Briefly, a FRET oligonucleotide probe is designed which overlaps theflanking and insert DNA junction. The FRET probe and PCR primers (oneprimer in the insert DNA sequence and one in the flanking genomicsequence) are cycled in the presence of a thermostable polymerase anddNTPs. Hybridization of the FRET probe results in cleavage and releaseof the fluorescent moiety away from the quenching moiety on the FRETprobe. A fluorescent signal indicates the presence of theflanking/transgene insert sequence due to successful amplification andhybridization.

Molecular Beacons have been described for use in sequence detection asdescribed in Tyangi et al. (Nature Biotech. 14:303-308, 1996). Briefly,a FRET oligonucleotide probe is designed that overlaps the flanking andinsert DNA junction. The unique structure of the FRET probe results init containing secondary structure that keeps the fluorescent andquenching moieties in close proximity. The FRET probe and PCR primers(one primer in the insert DNA sequence and one in the flanking sequence)are cycled in the presence of a thermostable polymerase and dNTPs.Following successful PCR amplification, hybridization of the FRET probeto the target sequence results in the removal of the probe secondarystructure and spatial separation of the fluorescent and quenchingmoieties. A fluorescent signal results. A fluorescent signal indicatesthe presence of the flanking/transgene insert sequence due to successfulamplification and hybridization.

A hybridization reaction using a probe specific to a sequence foundwithin the amplicon is yet another method used to detect the ampliconproduced by a PCR reaction.

Embodiments of the present invention are further defined in thefollowing Examples. It should be understood that these Examples aregiven by way of illustration only. From the above discussion and theseExamples, one skilled in the art can ascertain the essentialcharacteristics of this invention, and without departing from the spiritand scope thereof, can make various changes and modifications of theembodiments of the invention to adapt it to various usages andconditions. Thus, various modifications of the embodiments of theinvention, in addition to those shown and described herein, will beapparent to those skilled in the art from the foregoing description.Such modifications are also intended to fall within the scope of theappended claims.

The disclosure of each reference set forth herein is incorporated hereinby reference in its entirety.

EXAMPLES Example 1 Transformation of Maize by Agrobacteriumtransformation and Regeneration of Transgenic Plants Containing theCry34Ab1 and Cry35Ab1 (Cry34/35Ab1) Genes

A DNA molecule of approximately 7.4 Kb, designated PHI17662A (SEQ ID NO:24), which includes a first transgene expression cassette comprising aDNA molecule which includes the promoter, 5′ untranslated exon, andfirst intron of the maize ubiquitin (Ubi-1) gene (Christensen et al.(1992) Plant Mol. Biol. 18:675-689 and Christensen and Quail (1996)Transgenic Res. 5:213-218) operably connected to a DNA molecule encodinga B.t. δ-endotoxin identified as Cry34Ab1 (U.S. Pat. Nos. 6,127,180,6,624,145 and 6,340,593) operably connected to a DNA molecule comprisinga Pin II transcriptional terminator isolated from potato (Gyheung An etal. (1989) Plant Cell. 1:115-122). The second transgene expressioncassette of the DNA construct comprises a DNA molecule encoding thewheat peroxidase promoter (Hertig et al (1991) Plant Mol. Biol.16:171-174) operably connected to a DNA molecule encoding a B.t.δ-endotoxin identified as Cry35Ab1 (U.S. Pat. Nos. 6,083,499, 6,548,291and 6,340,593) operably connected to a DNA molecule comprising a Pin IItranscriptional terminator isolated from potato (Gyheung An et al.(1989) Plant Cell. 1:115-122). The third transgene expression cassetteof the DNA construct comprises a DNA molecule of the cauliflower mosaicvirus (CaMV) 35S promoter (Odell J. T. et al. (1985) Nature 313:810-812; Mitsuhara et al. (1996) Plant Cell Physiol. 37: 49-59) operablyconnected to a DNA molecule encoding a phosphinothricinacetyltransferase (PAT) gene (Wohlleben W. et al. (1988) Gene 70: 25-37)operably connected to a DNA molecule comprising a 3′ transcriptionalterminator from (CaMV) 35S (see Mitsuhara et al. (1996) Plant CellPhysiol. 37: 49-59) was used to transform maize embryo tissue.

B.t. Cry34/35 Ab1 maize plants were obtained by Agrobacteriumtransformation, the method of Zhao was employed (U.S. Pat. No.5,981,840, and PCT patent publication WO98/32326; the contents of whichare hereby incorporated by reference). Briefly, immature embryos wereisolated from maize and the embryos contacted with a suspension ofAgrobacterium, where the bacteria was capable of transferring PHI17662DNA (SEQ ID NO:24) to at least one cell of at least one of the immatureembryos (step 1: the infection step). Specifically, in this step theimmature embryos were immersed in an Agrobacterium suspension for theinitiation of inoculation. The embryos were co-cultured for a time withthe Agrobacterium (step 2: the co-cultivation step). Specifically, theimmature embryos were cultured on solid medium following the infectionstep. Following this co-cultivation period a “resting” step wasprovided. In this resting step, the embryos were incubated in thepresence of at least one antibiotic known to inhibit the growth ofAgrobacterium without the addition of a selective agent for planttransformants (step 3: resting step). In particular, the immatureembryos are cultured on solid medium with antibiotic, but without aselecting agent, for elimination of Agrobacterium and for a restingphase for the infected cells. Next, inoculated embryos were cultured onmedium containing a selective agent and growing transformed callus wasrecovered (step 4: the selection step). Specifically, the immatureembryos were cultured on solid medium with a selective agent resultingin the selective growth of transformed cells. The callus was thenregenerated into plants (step 5: the regeneration step), and,specifically, calli grown on selective medium were cultured on solidmedium to regenerate the plants. Individual embryos were kept physicallyseparate during culture, and the majority of explants died on theselective medium.

Those embryos that survived and produced healthy, glufosinate-resistantcallus tissue were assigned unique identification codes representingputative transformation events, and continually transferred to freshselection medium. Plants were regenerated from tissue derived from eachunique event and transferred to the greenhouse. Leaf samples were takenfor molecular analysis to verify the presence of the transgene by PCRand to confirm expression of the Cry34/35Ab1 protein by ELISA. Plantswere then subjected to a whole plant bioassay using western cornrootworm insects. Positive plants were crossed with inbred lines toobtain seed from the initial transformed plants. A number of lines wereevaluated in the field. The DAS-59122-7 event was selected from apopulation of independent transgenic events based on a superiorcombination of characteristics, including insect resistance andagronomic performance.

Example 2 Identification of Bacillus thuringiensis Cry34/35Ab1 MaizeLine DAS-59122-7

Seed from event DAS-59122-7 was evaluated. The T1S2 seed representstransformation into the Hi-II background, followed by a cross withinbred line PH09B and two rounds of self-crossing. All seed wereobtained from Pioneer Hi-Bred (Johnston, Iowa). Primary characterizationwas conducted on plant leaf tissue during the study by confirmation ofphosphinothricin acetyltransferase (PAT) activity via herbicide leafpainting and Cry34Ab1 expression using lateral flow devices.

Control substances in this study were defined as unmodified seedrepresentative of the test substance background. Control seeds of Hi-IIand PH09B backgrounds were used as negative controls. These unmodifiedseed do not contain the plant transcription units for the cry34Ab1,cry35Ab1, and pat genes. All seed were obtained from Pioneer Hi-Bred(Johnston, Iowa).

DNA samples from two additional B.t. Cry34/35Ab1 events, eventDAS-45214-4 and event DAS-45216-6, were used as negative controls forevent specific PCR analysis. The two events were produced throughAgrobacterium transformation using the same vector used to produce eventDAS-59122-7 and therefore contained the plant transcription units forthe cry34Ab1, cry35Ab1, and pat genes. However, the insertions sites ofthe T-DNA in events DAS-45214-4 and DAS-45216-6, including genomic DNAborder regions, were different from that in event DAS-59122-7. DNAsamples from event DAS-45214-4 and event DAS-45216-6 were isolated andcharacterized by Southern blot analysis. (Data not shown.) Corn seed forevent DAS-59122-7 and unmodified control seed (Hi-II and PH09B) wereplanted in growth chambers at the DuPont Experimental Station(Wilmington, Del.) to produce sufficient numbers of plants for DNAanalysis. For characterization of event DAS-59122-7, ten (10) T1S2 seedswere planted. Ten (10) seeds were also planted for each unmodifiedcontrol line. One (1) seed was planted per pot, and the pot was uniquelyidentified. Planting and growing conditions were conducive to healthyplant growth including regulated light and water.

Leaf samples were collected for each of the control and eventDAS-59122-7 plants. For each sample, sufficient leaf material from abovethe growing point was collected and placed in a pre-labeled sample bag.The samples were placed on dry ice and were transferred to an ultralowfreezer following collection. All samples were maintained frozen untilprocessing. All leaf samples were uniquely labeled with the plantidentifier and the date of harvest.

To confirm the expression of the Cry34Ab1 protein in event DAS-59122-7and the absence of expression in the controls, leaf samples werecollected from all event DAS-59122-7 and control plants, and screenedfor transgenic protein using lateral flow devices specific for Cry34Ab1(Strategic Diagnostics, Inc., Newark, Del.). Leaf punches were takenfrom each plant and ground in a phosphate buffered saline solution withTween 20 to crudely extract the protein. A strip device was dipped intothe extract to determine the presence or absence of the Cry34Ab1protein. The immunoassay results were used to confirm the identity ofthe test substance plants prior to molecular analysis as shown in Table1.

To confirm the expression of phosphinothricin acetyltransferase (PAT) inevent DAS-59122-7 plants, herbicide leaf painting was conducted. Allplants used in this study were leaf painted to confirm plant identity.Plants were assayed prior to the R1 growth stage. Assays were conductedfollowing a standard procedure known in the art for herbicide leafpainting for the identification of PAT-expressing transgenic plants.Specifically, a portion of one leaf of each plant was treated withapproximately 2% solution of glufosinate herbicide, Basta® (BayerCropScience) in water and visually checked for brown or necrotic tissuein the painted leaf area 4-12 days after application. Results for eachplant were recorded and used to determine expression of PAT in each testplant as shown in Table 1. As shown in Table 1, of the ten (10) plantstested for event DAS-59122-7 T1S2 generation, six (6) plants expressedboth Cry34Ab1 and PAT, while four (4) plants did not express eitherprotein. All unmodified controls tested negative for both CryAb1 And PATassays (data not shown).

TABLE 1 Cry34Ab1 and PAT Protein Expression and Southern HybridizationData for B.t. Cry34/35Ab1 Event DAS-59122-7 Southern Southern SouthernCry34Ab1 Blot Blot Blot and PAT cry34Ab1 cry35Ab1 pat Plant ID Sample IDExpression¹ Probe² Probe² Probe² 02-122C 1 DAS59122-7 positive + + +T1S2 1 02-122C 2 DAS59122-7 positive + + + T1S2 2 02-122C 3 DAS59122-7positive + + + T1S2 3 02-122C 4 DAS59122-7 negative − − − T1S2 4 02-122C5 DAS59122-7 positive + + + T1S2 5 02-122C 6 DAS59122-7 negative − − −T1S2 6 02-122C 7 DAS59122-7 positive + + + T1S2 7 02-122C 8 DAS59122-7negative − − − T1S2 8 02-122C 9 DAS59122-7 negative − − − T1S2 9 02-122C10 DAS59122-7 positive + + + T1S2 10 ¹Positive Cry34Ab1 expressionindicates detection of protein expression as determined by theimmunoassay-based lateral flow device specific for Cry34Ab1 proteindetection. Negative indicates no detection of the Cry34Ab1 protein.Positive PAT expression indicates plants that were tolerant to theherbicide treatment and negative indicates plants that were sensitive tothe herbicide. ²+ indicates hybridization signal on Southern blot; −indicates no hybridization signal on Southern blot. The cry34Ab1 geneprobe hybridized to the expected internal T-DNA fragment of 1.915 kb,the cry35Ab1 gene probe hybridized to the expected internal T-DNAfragment of 2.607 kb, and the pat gene probe hybridized to a 3.4 kbborder fragment consistent with a single intact T-DNA insertion asdetermined by Southern blot analysis.

Example 3 Southern Blot Analysis of Bacillus thuringiensis Cry34/35Ab1Maize Line DAS-59122-7

One gram quantities of leaf samples were ground under liquid nitrogen,and the genomic DNA was isolated using DNeasy Plant Mini Kit (Qiagen,Valencia, Calif.) or using a standard Urea Extraction Buffer procedure.Following extraction, the DNA was visualized on an agarose gel todetermine the DNA quality, and was quantified using Pico Green® reagent(Molecular Probes, Inc., Eugene, Oreg.) and spectrofluorometricanalysis.

The 1 Kb DNA Ladder (Invitrogen, Carlsbad, Calif.) was used to estimateDNA fragment sizes on agarose gels.

Genomic DNA isolated from event DAS-59122-7 plants was digested with NcoI and electrophoretically separated, transferred to nylon membranes, andhybridized to the cry34Ab1, cry35Ab1 and pat gene probes using standardprocedures known in the art. Blots were exposed to X-ray film for one ormore time periods to detect hybridizing fragments and to visualizemolecular weight standards. Images were then digitally captured byphotographing X-ray films and/or by detection with a Lumi-Imager™instrument (Roche, Indianapolis, Ind.). The sizes of detected bands weredocumented for each probe. Southern blot analysis was used as a means ofverifying the presence of the insertion in the test plants andconfirming that all plants from event DAS-59122-7 contained the sameinsertion as shown in Table 1. (Southern blots not shown.) Southern blotanalysis indicated that event DAS-59122-7 contained a single insertionconsisting of an intact copy of the T-DNA region from plasmid PHP17662,while the null segregants, as determined by the protein expressionanalysis did not hybridize to the gene probes. Further, eventDAS-59122-7 plants expressing the two proteins exhibited identicalhybridization patterns on Southern blots (data not shown). Specifically,the cry34Ab1 gene probe hybridized to the expected internal T-DNAfragment of 1.915 kb, the cry35Ab1 gene probe hybridized to the expectedinternal T-DNA fragment of 2.607 kb, and the pat gene probe hybridizedto a 3.4 kb border fragment consistent with a single intact T-DNAinsertion as determined by Southern blot results.

Example 4 T-DNA Insert and Flanking Border Region Sequencing of Bacillusthuringiensis Cry34/35Ab1 Maize Line DAS-59122-7

The T-DNA insert and flanking border regions were cloned from B.t.Cry34/35Ab1 event DAS59122-7 using PCR based methods as diagramed inFIGS. 2 and 3. Specifically, sequences bordering the 5′ and 3′ ends ofthe insert in event DAS-59122-7 were obtained using two genome walkingtechniques. The first walking method was essentially the method asdescribed for the Universal Genome Walker Kit (BD Biosciences Clontech,Palo Alto, Calif.), and the second method was conducted according to thesplinkerette protocol outlined in Devon et al., (1995) Nucleic AcidsResearch 23 (9):1644-1645, with modifications as described by Stover(2001), U. C. Irvine (personal communication).

Briefly, genomic DNA was digested with various restriction enzymes (DraI, EcoR V, Pvu II, Sma I and Stu I for the Universal Genome Walkermethod and BamH I, EcoR I, Hind III, and Xba I for the splinkerettemethod) and then ligated to blunt-end adaptors for the Genome Walkermethod and to adaptors specific for the restriction enzyme used for thesplinkerette method. The adaptors for both genome walking methods weredesigned to prevent extension of the 3′ end of the adaptor during PCRand thus reduce or eliminate nonspecific amplification. Theadaptor-ligated genomic DNA fragments were then referred to as genomewalker libraries or splinkerette libraries, one library for eachrestriction enzyme. Libraries were prepared from genomic DNA isolatedfrom three individual T1S2 plants of B.t. Cry34/35Ab1 event DAS-59122-7;plants DAS-59122-7 T1S2 1, DAS-59122-7 T1S2 2 and DAS-59122-7 T1S2 10,and from one Hi-II and one PH09B control plant.

Following construction of the libraries, nested PCR amplifications werecompleted to amplify the target sequence using Advantage™-GC Genomic PCRkit (BD Biosciences Clontech, Palo Alto, Calif.). The primary PCRamplification used one primer with identity to the adaptor and one genespecific primer. The adaptor primer will not amplify a product in thefirst cycle of the primary PCR and only products from the gene specificprimer will be produced. Annealing and amplification from the adaptorprimer only occurs after the complementary strand has been produced fromthe gene specific primer. Following primary PCR amplification, asecondary nested PCR reaction was performed to increase the specificityof the genomic PCR reactions. The nested primers consisted ofgene-specific and adaptor-specific sequences internal to the respectiveprimers used in the primary PCR.

For 5′ flanking border sequences, nested PCR was initiated using primersspecific to the 5′ end of the inserted T-DNA along with primerscomplementary to the adaptor sequence ligated onto the digested DNA.Similarly, cloning of the 3′ flanking border sequence started with aprimer specific for the 3′ end of the inserted T-DNA and a primercomplementary to the adaptor sequence. DNA sequences internal to theT-DNA Right Border and Left Border sequences within the T-DNA regionwere used as the starting points for “walking out” to the maize genomicsequence, because they represented unique sequence (not homologous toendogenous maize genomic sequences) from which to anchor the genomewalking primers.

The products produced by the nested PCR were analyzed by agarose gelelectrophoresis (data not shown). Fragments visible in librariesprepared from one or more of the event DAS-59122-7 DNA samples andabsent in libraries prepared from the Hi-II and PH09B genomic DNAsamples were identified for further characterization. The identified PCRamplified fragments were separated by preparatory gel electrophoresis,isolated using a QIAquick Gel Extraction Kit (Qiagen), and sent directlyfor sequencing or cloned into a pGEM-T Easy plasmid vector using thepGEM-T Easy Vector System I (Promega Corp., Madison, Wis.) prior to DNAsequencing. Sequencing reactions were carried out with primers used forthe nested PCR amplification or with primers specific for use with thepGEM-T Easy vector. The sequence obtained was used to design additionalgene specific primers to continue “walking out” into the unknown maizegenomic sequence. Multiple rounds of genome walking were performed untilat least 500 bp of border sequence from the ends of the T-DNA insertwere obtained.

To ensure validity of the flanking border sequences, additionalevent-specific PCR amplifications on genomic DNA from event DAS-59122-7were performed. The amplified fragments were sequenced in order tofurther extend the region of sequence overlap from the T-DNA insertregion into the 5′ and 3′ bordering genomic DNA. Primers, shown in Table2, were designed based on the sequence obtained from the genome walkingexperiments to amplify a fragment spanning the unique junction of theT-DNA with the corn genomic DNA. Primer set 03-O-506/02-O-476 (SEQ IDNO: 10/SEQ ID NO:9) spanned the 5′ junction and amplified a 313 bpfragment (from bp 2427 to bp 2739, see FIG. 1), and primer set02-O-447/03-O-577 (SEQ ID NO: 8/SEQ ID NO:17) spanned the 3′ junctionand amplified a 754 bp fragment (from bp 9623 to bp 10376, see FIG. 1).

TABLE 2 Primer Sequences Target Sequence Primer Location Name Sequence(5′-3′) (bp to bp)¹ 02-O-215 (SEQ ID NO: 1) 2743-2716GTGGCTCCTTCAACGTTGCGGTTCTG TC 02-O-219 (SEQ ID NO: 2) 9830-9858CGTGCAAGCGCTCAATTCGCCCTATA GTG 02-O-227 (SEQ ID NO: 3) 9846-9827AATTGAGCGCTTGCACGTTT 02-O-370 (SEQ ID NO: 4) 4871-4894AACAACAAGACCGGCCACACCCTC 02-O-371 (SEQ ID NO: 5) 5187-5163GAGGTGGTCTGGATGGTGTAGGTCA 02-O-372 (SEQ ID NO: 6) 7017-7044TACAACCTCAAGTGGTTCCTCTTCCC GA 02-O-373 (SEQ ID NO: 7) 7897-7873GAGGTCTGGATCTGCATGATGCGGA 02-O-447 (SEQ ID NO: 8) 9623-9645AACCCTTAGTATGTATTTGTATT 02-O-476 (SEQ ID NO: 9) 2739-2714CTCCTTCAACGTTGCGGTTCTGTCAG 03-O-506 (SEQ ID NO: 10) 2427-2451TTTTGCAAAGCGAACGATTCAGATG 03-O-514 (SEQ ID NO: 11) 2687-2709GCGGGACAAGCCGTTTTACGTTT 03-O-542 (SEQ ID NO: 12) 1076-10742GACGGGTGATTTATTTGATCTGCAC 03-O-543 (SEQ ID NO: 13) 2451-2427CATCTGAATCGTTCGCTTTGCAAAA 03-O-564 (SEQ ID NO: 14) 2324-2299CTACGTTCCAATGGAGCTCGACTGTC 03-O-569 (SEQ ID NO: 15) 10150-10174GGTCAAGTGGACACTTGGTCACTCA 03-O-570 (SEQ ID NO: 16) 10275-10299GAGTGAAGAGATAAGCAAGTCAAAG 03-O-577 (SEQ ID NO: 17) 10376-10352CATGTATACGTAAGTTTGGTGCTGG 03-O-784 (SEQ ID NO: 18) 2189-2213AATCCACAAGATTGGAGCAAACGAC 67609 (SEQ ID NO: 36) 9862-9886CGTATTACAATCGTACGCAATTCAG 69240 (SEQ ID NO: 37) 9941-9965GGATAAACAAACGGGACCATAGAAG ¹Location in sequence of Event DAS-59122-7(see FIG. 1). Bases 1 − 2593 = 5′ border, bases 2594 − 9936 = T-DNAinsert, bases 9937 − 11922 = 3′ border.

For verification of the DNA sequence that inserted into the maizegenome, PCR was performed to amplify, clone, and sequence the insertedT-DNA from event DAS-59122-7. PCR primer sets, (SEQ ID NO: 11/SEQ IDNO:5); (SEQ ID NO: 4/SEQ ID NO:7); and (SEQ ID NO: 6/SEQ ID NO:3) shownin Table 3 were used to amplify three overlapping fragments labeled22I-1 (SEQ ID NO: 25), 22I-2 (SEQ ID NO: 26), and 22I-3 (SEQ ID NO: 27)representing sequence from the 5′ region of the T-DNA running through tothe 3′ region of the T-DNA insert from bp 2687 to bp 9846 for eventDAS-59122-7 (see FIG. 1). PCR amplicon information is reported in Table3 and primer sequences are listed in Table 2.

TABLE 3 PCR Primer and Amplicon Descriptions Location of PCR PCR SizeTarget Forward Reverse Amplicon Amplicon (bp) Sequence Primer Primer (bpto bp)¹ 22I-1 2501 T-DNA 03-O-514 02-O-371 2687-5187 (SEQ ID insert (SEQID (SEQ ID NO: 25) NO: 11) NO: 5) 22I-2 3027 T-DNA 02-O-370 02-O-3734871-7897 (SEQ ID insert (SEQ ID (SEQ ID NO: 26) NO: 4) NO: 7) 22I-32830 T-DNA 02-O-372 02-O-227 7017-9846 (SEQ ID insert (SEQ ID (SEQ IDNO: 27) NO: 6) NO: 3) O784/O564 136 5′ genomic 03-O-784 03-O-5642189-2324 (SEQ ID border (SEQ ID (SEQ ID NO: 28) NO: 18) NO: 14)O784/O543 263 5′ genomic 03-O-784 03-O-543 2189-2451 (SEQ ID border (SEQID (SEQ ID NO: 29) NO: 18) NO: 13) O569/O577 227 3′ genomic 03-O-56903-O-577 10150-10376 (SEQ ID border (SEQ ID (SEQ ID NO: 30) NO:15) NO:17) O570/O542 492 3′ genomic 03-O-570 03-O-542 10275-10766 (SEQ IDborder (SEQ ID (SEQ ID NO: 31) NO: 16) NO: 12) O784/O215 555 5′ 03-O-78402-O-215 2189-2743 (SEQ ID junction (SEQ ID (SEQ ID NO: 32) NO: 18)NO: 1) O219/O577 547 3′ 02-O-219 03-O-577  9830-10376 (SEQ ID junction(SEQ ID (SEQ ID NO: 33) NO: 2) NO: 17) O506/O476 313 5′ 03-O-50602-O-476 2427-2739 (SEQ ID junction (SEQ ID (SEQ ID NO: 34) NO: 10) NO:9) O447/O577 754 3′ 02-O-447 03-O-577  9623-10376 (SEQ ID junction (SEQID (SEQ ID NO: 35) NO: 8) NO: 17) 67609/69240 104 3′ 67609 692409862-9965 (SEQ ID junction (SEQ ID (SEQ ID NO: 38) NO: 36) NO: 37)¹Location in sequence of Event DAS-59122-7 (see FIG. 1). Bases 1-2593 =5′ border, bases 2594-9936 = T-DNA insert, bases 9937-11922 = 3′ border.

PCR GC2 Advantage™ Polymerase kit (BD Biosciences Clontech, Inc.) wasused according to manufacturer's instructions to amplify the insertfragments (22I-1 (SEQ ID NO: 25), 22I-2 (SEQ ID NO: 26), and 22I-3 (SEQID NO: 27)). Briefly, a 50 L reaction contained 5′ and 3′ primers at afinal concentration of 0.2 μM and 40 ng of genomic DNA. PCR reactionswere set up in duplicate using genomic DNA preparation from plantsDAS-59122-7 T1S2 1 and DAS-59122-7 T1S2 2. PCR conditions were asfollows: initial denaturation at 95° C. for 1 min, followed by 35 cyclesof 94°/95° C. for 30 sec, 55° C. for 30 sec, and 68° C. for 5 min, withfinal extension at 68° C. for 6 min. PCR amplification products werevisualized under UV light, following electrophoresis through a 1%agarose gel in 1×TBE (89 mM Tris-Borate, 2 mM EDTA, pH 8.3) stained withethidium bromide.

PCR fragments 22I-1 (SEQ ID NO: 25 ), 22I-2 (SEQ ID NO: 26), and 22I-3(SEQ ID NO: 27) were purified by excising the fragments from 0.8%agarose gel in 1×TBE stained with ethidium bromide, and purifying thefragment from the agarose using a QIAquick Gel Extraction Kit (Qiagen).PCR fragments were cloned into a pGEM-T Easy plasmid vector using thepGEM-T Easy Vector System I (Promega Corp.). Cloned fragments wereverified by minipreparation of the plasmid DNA (QIAprep Spin MiniprepKit, Qiagen) and restriction digestion with Not I. Plasmid clones and/orpurified PCR insert fragments were then sent for sequencing of thecomplete insert. Sequencing reactions were carried with primers designedto be specific for known T-DNA sequences or with primers specific foruse with the pGEM-T Easy vector. Sigma-Genosys, Inc. (The Woodlands,Tex.) synthesized all PCR primers, which were used at a finalconcentration of 0.2-0.4 μM in the PCR reactions.

PCR reactions with genomic DNA isolated from B.t. Cry34/35Ab1 eventsDAS-59122-7, DAS-45214-4, and DAS-45216-6, and unmodified control linesHi-II and PH09B were used to confirm (1) the presence of maize genomicDNA in the sequenced border regions of event DAS-59122-7, and (2) eventspecific amplification across the junctions of the T-DNA insert andgenomic DNA borders in event DAS-59122-7.

PCR primers designed to amplify the border sequence flanking the insertin event DAS-59122-7 were used to confirm the presence of those regionsin unmodified control lines as well as in event DAS-59122-7. Two (2)sets of primers each, for the 5′ and 3′ borders (four (4) sets total)were tested. Primer sets 03-O-784/03-O-564 (SEQ ID NO: 18/SEQ ID NO:14)and 03-O-784/03-O-543 (SEQ ID NO: 18/SEQ ID NO: 13) were used to amplify136 bp and 263 bp fragments, respectively, from border sequence 5′ tothe T-DNA insert in event DAS-59122-7 (FIGS. 2 and 3). Similarly, primersets 03-O-569/03-O-577 (SEQ ID NO: 15/SEQ ID NO: 17) and03-O-570/03-O-542 (SEQ ID NO: 16/SEQ ID NO: 12) were used to amplify 227bp and 492 bp fragments, respectively, from the 3′ genomic border (FIGS.2 and 3).

Primers designed to amplify fragments across the junction of the bordersequence and T-DNA insert were used to establish event-specific PCRfragments for event DAS-59122-7. One primer set was selected for each ofthe two junctions. Primer set 03-O-784/02-O-215 (SEQ ID NO: 18/SEQ IDNO:1) was designed to amplify a 555 bp fragment across the 5′ junction,and primer set 02-O-219/03-O-577 (SEQ ID NO: 2/SEQ ID NO: 17) wasdesigned for amplification of a 547 bp fragment at the 3′ junction. Aset of primers, IVR1(0197) (SEQ ID NO: 39)5′-CCGCTGTATCACAAGGGCTGGTACC-3′ and IVR2(0198) (SEQ ID NO: 40)5′-GGAGCCCGTGTAGAGCATGACGATC-3′, based on the endogenous maize invertasegene (Hurst et al., (1999) Molecular Breeding 5 (6):579-586), was usedto generate a 226 bp amplification product as an internal positivecontrol for all maize genomic DNA samples.

All PCR primers were synthesized by Sigma-Genosys, Inc. and used at afinal concentration of 0.2-0.4 μM in the PCR reactions. PCR primersequences are listed in the Table 2. For PCR amplifications,Advantage™-GC 2 PCR kit (BD Biosciences) was used according tomanufacturer's instructions. Approximately 10-100 ng of genomic DNAtemplate was used per 50 μL PCR reaction. PCR conditions were asfollows: initial template denaturation at 94° C. for 5 min, followed by35 cycles of 95° C. for 1 minute, 60° C. for 2 minutes, and 72° C. for 3min, with final extension at 72° C. for 7 min. The PCR amplificationproducts were visualized under UV light following electrophoresisthrough a 1% agarose gel with 1×TBE and ethidium bromide.

Sequence data obtained for the T-DNA insert and border regions of eventDAS-59122-7 was reviewed and assembled using Seqman II™ software Version4.0.5 (DNAStar, Inc., Madison, Wis.). The 5′ and 3′ border sequencesflanking the insert present in event DAS-59122-7 were used for homologysearching against the GenBank public databases in order to furthercharacterize the site of insertion in the maize genome. Analysis toidentify open reading frames in the junction regions between theflanking borders and T-DNA insert in event DAS-59122-7 was conductedusing Vector NTI 8.0 (InforMax™, Inc., Frederick, Md.).

In total, 11922 bp of sequence from genomic DNA of event DAS-59122-7 wasconfirmed (see FIG. 1). At the 5′ end of the T-DNA insert, 2593 bp offlanking border sequence was identified, and 1986 bp of flanking bordersequence was obtained on the 3′ end from fragments derived from genomewalking experiments. A total of 7160 bp of the T-DNA insert was clonedand sequenced using PCR primer sets designed to amplify threeoverlapping fragments labeled 22I-1 (2501 bp) (SEQ ID NO:25), 22I-2(3027 bp) (SEQ ID NO:26), and 22I-3 (2830 bp) (SEQ ID NO:27)representing sequence from the 5′ region of the T-DNA running through tothe 3′ region of the T-DNA insert for event DAS-59122-7 from bp 2687 tobp 9846 (see FIG. 1). The remainder of the T-DNA insert region wassequenced from two PCR fragments, 0506/0476 (SEQ ID NO: 10/SEQ ID NO:9)and 0447/0577 (SEQ ID NO: 8/SEQ ID NO: 17) that spanned the 5′ and 3′junctions, respectively, of the T-DNA insert with corn genomic DNA.Primers used were designed based on the sequence obtained from thegenome walking experiments to amplify a fragment spanning the uniquejunction of the T-DNA with the corn genomic DNA. Primer set03-O-506/03-O-476 (SEQ ID NO: 10/SEQ ID NO:9) spanned the 5′ junctionand amplified a 313 bp fragment (from bp 2427 to bp 2739) and primer set03-O-447/03-O-577 (SEQ ID NO: 8/SEQ ID NO:17) spanned the 3′ junctionand amplified a 754 bp fragment (from bp 9623 to bp 10376). Combined, atotal of 7343 bp of the T-DNA insert in event DAS-59122-7 was cloned andsequenced (from bp 2594 to bp 9936, see FIG. 1) and compared to thesequence of the transforming plasmid, PHP17662. Two nucleotidedifferences at bp 6526 and bp 6562 were observed in the non-translatedwheat peroxidase promoter region of the T-DNA insert (see FIG. 1).Neither of the observed base changes affected the open reading framecomposition of the T-DNA insert. Both the 3′ and 5′ end regions of theT-DNA insert were found to be intact, except for deletion of the last 22bp at the 5′ end and 25 bp at the 3′ end encompassing the Right and LeftT-DNA Border regions, respectively. While T-DNA border sequences areknown to play a critical role in T-DNA insertion into the genome, thisresult is not unexpected since insertions are often imperfect,particularly at the Left T-DNA Border (Tinland (1996) Trends in PlantScience 1(6):178-184).

BLAST (Basic Local Alignment Search Tool) analysis of the genomic borderregions of event DAS-59122-7 showed limited homology with publiclyavailable sequences (Release 138.0 GenBank, Oct. 25, 2003). Analysis ofthe 5′ border region found two areas with significant homology to maizegenomic and EST (Expressed Sequence Tag) sequences. The first areaencompasses 179 bp (bp 477 to bp 655 of the border sequence) anddisplays similarity to several molecular markers, chromosomal sequences,and consensus sequences obtained by alignment of various ESTs. Thesecond area occurs at bp 1080 to bp 1153 (74 bp) of the 5′ bordersequence, and shows similarity to a number of different maize ESTs andgenomic sequences. The 3′ border region also had two smallnon-contiguous regions of similarity to plant DNA sequences. The inner3′ region of 162 bp (bp 9954 to bp 10115) showed similarity to the 3′untranslated end of two genomic Zea mays alcohol dehydrogenase (adh1)genes as well as to several EST consensus sequences. A smaller region(57 bp) in the middle of the 3′ border (bp 10593 to bp 10649) showedsimilarity to non-coding regions from multiple maize genomic sequences.

Overall, no homologous regions greater than 179 base pairs wereidentified in either of the genomic border sequences, nor was more thanone homologous region from the same known sequence found. Individualaccessions displaying similarity to the event DAS-59122-7 bordersequences were examined to determine if the insertion in eventDAS-59122-7 occurred in a characterized protein coding sequence. None ofthe regions of similarity occurred within any known protein codingsequences. Local alignment of the entire transformation plasmidsequence, PHP17662, with the event DAS-59122-7 border sequences showedno significant homologies, indicating that the border regions flankingthe T-DNA insert did not contain fragments of the transforming plasmid.Therefore, identification and characterization of the genomic sequenceflanking the insertion site in event DAS-59122-7 was limited due to theabsence of significant regions of homology to known sequences.

The 5′ and 3′ junction regions between the maize genomic border sequenceand the T-DNA insert in event DAS-59122-7 were analyzed for the presenceof novel open reading frames. No open reading frames of significant size(>100 amino acids) were identified in the 5′ or 3′ border junctionregions, indicating that no novel open reading frames were generated asa result of the T-DNA insertion. Additionally, the homology searches didnot indicate the presence of endogenous maize open reading frames in theborder regions that might have been interrupted by the T-DNA insertionin B.t. Cry34/35Ab1 event DAS-59122-7.

Example 5 PCR Primers

DNA event specific primer pairs were used to produce an amplicondiagnostic for DAS-59122-7. These event primer pairs include, but arenot limited to, SEQ ID NO: 18 and SEQ ID NO: 1; SEQ ID NO: 2 and SEQ IDNO: 17; SEQ ID NO: 10 and SEQ ID NO: 9; and SEQ ID NO: 8 and SEQ ID NO:17; and SEQ ID NO: 36 and SEQ ID NO: 37. In addition to these primerpairs, any primer pair derived from SEQ ID NO: 21 and SEQ ID NO: 22 thatwhen used in a DNA amplification reaction produces a DNA amplicondiagnostic for DAS-59122-7 is an embodiment of the present invention.Any modification of these methods that use DNA primers or complementsthereof to produce an amplicon DNA molecule diagnostic for DAS-59122-7is within the ordinary skill of the art. In addition, control primerpairs, which include IVR1(0197)/IVR2(0198) (SEQ ID NO: 39/SEQ ID NO: 40)for amplification of an endogenous corn gene are included as internalstandards for the reaction conditions.

The analysis of plant tissue DNA extracts to test for the presence ofthe DAS-59122-7 event should include a positive tissue DNA extractcontrol (a DNA sample known to contain the transgenic sequences). Asuccessful amplification of the positive control demonstrates that thePCR was run under conditions that allow for the amplification of targetsequences. A negative, or wild-type, DNA extract control in which thetemplate DNA provided is either genomic DNA prepared from anon-transgenic plant, or is a non-DAS-59122-7 transgenic plant, shouldalso be included. Additionally a negative control that contains notemplate corn DNA extract will be a useful gauge of the reagents andconditions used in the PCR protocol.

Additional DNA primer molecules of sufficient length can be selectedfrom SEQ ID NO: 21 and SEQ ID NO: 22 by those skilled in the art of DNAamplification methods, and conditions optimized for the production of anamplicon diagnostic for event DAS-59122-7. The use of these DNA primersequences with modifications to the methods shown in these Examples arewithin the scope of the invention. The amplicon wherein at least one DNAprimer molecule of sufficient length derived from SEQ ID NO: 21 and SEQID NO: 22 that is diagnostic for event DAS-59122-7 is an embodiment ofthe invention. The amplicon wherein at least one DNA primer ofsufficient length derived from any of the genetic elements of PHI17662Athat is diagnostic for event DAS-59122-7 is an embodiment of theinvention. The assay for the DAS-59122-7 amplicon can be performed byusing a Stratagene Robocycler, MJ Engine, Perkin-Elmer 9700, orEppendorf Mastercycler Gradient thermocycler, or by methods andapparatus known to those skilled in the art.

Having illustrated and described the principles of the presentinvention, it should be apparent to persons skilled in the art that theinvention can be modified in arrangement and detail without departingfrom such principles. We claim all modifications that are within thespirit and scope of the appended claims.

All publications and published patent documents cited in thisspecification are incorporated herein by reference to the same extent asif each individual publication or patent application was specificallyand individually indicated to be incorporated by reference.

1. A method of detecting the presence of maize event DAS-59122-7 orprogeny thereof in a biological sample, comprising: (a) extracting a DNAsample from said biological sample; (b) providing a pair of DNA primermolecules selected from the group consisting of: i) the sequences of SEQID NO: 18 and SEQ ID NO: 1; ii) the sequences of SEQ ID NO:10 and SEQ IDNO:9; iii) the sequences of SEQ ID NO:2 and SEQ ID NO:17; and iv) thesequences of SEQ ID NO:8 and SEQ ID NO:17; (c) providing DNAamplification reaction conditions; (d) performing said DNA amplificationreaction, thereby producing a DNA amplicon molecule; and (e) detectingsaid DNA amplicon molecule, wherein the detection of said DNA ampliconmolecule in said DNA amplification reaction indicates the presence ofmaize event DAS-59122-7.